DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 07/01/2026 has been entered.
Applicant’s amendments to the claims and arguments filed on July 1, 2026 have been received and entered. Claim 50 has been amended, while claims 1-49, 51-52 have been canceled. Claims 50, 53-60 and 61 are pending in the instant application.
Election/Restrictions
Applicant’s election of SEQ ID NO: 16, 21 and cancer as species of at least one ribonucleic acid and disorder in the reply filed on October 3, 3025 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Priority
This application is a Divisional of US application no 15/524,968 05/05/2017, which is a 371 of PCT/US15/59621 filed on 11/06/2015 that claims priority from US provisional application no 62/076,424 filed on 11/06/2014. The disclosure of the prior-filed US provisional application No. 62/076424, fails to provide adequate support or enablement in the manner provided by the first paragraph of 35 U.S.C. 112 for one or more claims of this application. Instant claims 50 and 55 requires (i) at least one ribonucleic acid selected from the group consisting of SEQ ID NOs: 9-23 and 419-2609 (claim 50) and (ii) Cas protein comprising a Cpf1 protein or a functional portion thereof (claim 55). The ‘424 application discloses some of the gRNA sequence and a Cas protein but does not describe using several of the gRNA sequence (for example SEQ ID NOs: 419-2609) and Cpf1 as Cas protein as instantly claimed. Consequently, there is no written description in application for the use of different gRNA sequence and Cpf1 in ‘424 application. In case if applicants have evidence to support otherwise, applicants are invited to indicate page and line number for the written support specifically for the claimed gRNA and Cpf1 as recited in claim 50 and 55 respectively of the instant application. Therefore, the effective filing date for instant claim 50, 53-55, 56, 58-61 is 11/06/2015, while the subject matter of claims 57 was described in the ‘424 application.
Claims 50, 53-60 and 61 are under consideration.
New- Claim Rejections - 35 USC § 112-in modified form
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 50, 53-60 and 61 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for: A method of engrafting an allogeneic CD34+ hematopoietic stem cells to a mouse in need, the method comprising:
(a) altering a target CCR5 polynucleotide sequence in a population of CD34+ hematopoietic stem cells ex vivo by contacting the CCR5 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated-9 (Cas 9) protein and at least one ribonucleic acid selected from group consisting of SEQ ID: 2-8, wherein the ribonucleic acid directs Cas protein to and hybridizes to a target motif of the target CCR5 polynucleotide sequence,
(b) altering a target B2M polynucleotide sequence in the population of CD34+ hematopoietic stem cells ex vivo by contacting the target B2M polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated-9 (Cas9) protein and at least one ribonucleic acid selected from the group consisting of SEQ ID NOs: 16 or 21 and 419-2609 to obtain a population of genetically modified CD34+ HSC comprising a genome in which B2M and CCR5 gene has been cleaved thereby eliminating surface expression of MHC1 molecule in the population of CD34+ hematopoietic stem cells; and
(c) administering the allogeneic population of genetically modified CD34+ hematopoietic stem comprising a genome in which B2M and CCR5 gene have been edited into the mouse, thereby engrafting CD34+ HSC in the subject in need thereof,
does not reasonably provide enablement for a method of administering any other cell to a subject having HIV or AIDS intended for treating HIV infection and/or treatment of AIDS, using any other Cas protein, using any other combination of gRNA targeting CCR5 and/or B2M, a xenogeneic transplant or using any other genetically modified cells.
The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
In determining whether Applicant's claims are enabled, it must be found that one of skill in the art at the time of invention by applicant would not have had to perform "undue experimentation" to make and/or use the invention claimed. Such a determination is not a simple factual consideration, but is a conclusion reached by weighing at least eight factors as set forth in In re Wands, 858 F.2d at 737, 8 USPQ 1400, 2d at 1404. Such factors are: (1) The breadth of the claims; (2) The nature of the invention; (3) The state of the art; (4) The level of one of ordinary skill in the art; (5) The level of predictability in the art; (6) The amount of direction and guidance provided by Applicant; (7) The existence of working examples; and (8) The quantity of experimentation needed to make and/or use the invention.
The office has analyzed the specification in direct accordance to the factors outlines in In re Wands. MPEP 2164.04 states: "[W]hile the analysis and conclusion of a lack of enablement are based on factors discussed in MPEP 2164.01(a) and the evidence as whole, it is not necessary to discuss each factor in written enablement rejection." These factors will be analyzed, in turn, to demonstrate that one of ordinary skill in the art would have had to perform "undue experimentation" to make and/or use the invention and therefore, applicant's claims are not enabled.
Nature of the Invention:
The claims are directed to a method of administering cells to a subject in need of such cells, the method comprising:(a) altering a target CCR5 polynucleotide sequence in a cell or a population of cells ex vivo by contacting the CCR5 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and at least one ribonucleic acid, wherein the ribonucleic acid directs Cas protein to and hybridizes to a target motif of the target CCR5 polynucleotide sequence, wherein the target polynucleotide sequence associated with the disorder is cleaved;(b) altering a target B2M polynucleotide sequence in the cell or population of cells ex vivo by contacting the target B2M polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and at least one ribonucleic acid selected from the group consisting of SEQ ID NOs: 9-23 and 419-2609; and(c) administering the cell or population of cells into the subject, wherein the subject has HIV or AIDs.. Claim 53 is directed to a method of claim 50 further comprising altering one or more additional polynucleotide sequences in the cell ex vivo. Claims 54-55 limit the Cas protein comprises a Cas9 protein or a functional portion thereof or a Cpfl protein. Claim 56 limits the cell or population of cells are selected from the group consisting of a stem cell, a pluripotent cell, a progenitor cells, a hematopoietic stem and/or progenitor cell, a CD34+ mobilized peripheral blood cell, a CD34+ cord blood cell, a CD34+ bone marrow cell, a CD34+CD38-Lineage- CD90+CD45RA- cell, and a CD34+ hematopoietic stem and/or progenitor cell; a CD4+ T cell, a hepatocyte, a somatic cell, and a non-transformed cell. Claim 58 limit the method of claim 50, wherein the alteration of the target B2M polynucleotide sequence in the cell or population of cells results in the deletion of a contiguous stretch of genomic DNA, thereby eliminating surface expression of MHC Class I molecules in the cell or population of cells.
Breadth of the claims:
The breadth of the claimed invention encompasses administering any genetically modified cells to a subject in need of such cells. The claims are broadly directed to (a) altering ay target CCR5 polynucleotide sequence in any cell or any population of cells ex vivo by contacting the polynucleotide sequence with any Cas protein and at least one ribonucleic acid, wherein the ribonucleic acid directs Cas protein to and hybridizes to a target motif of the target in any part of CCR5 polynucleotide sequence, wherein the CCR5 target polynucleotide sequence is cleaved; (b) altering a target B2M polynucleotide sequence in the cell or population of cells ex vivo by contacting the target B2M polynucleotide sequence with any Cas protein and at least one ribonucleic acid selected from the group consisting of SEQ ID NOs: 9-23 and 419-2609 and (c) introducing the cell or population of cells into the subject in need thereof such cells.
The specification broadly discusses cells of the invention to be used in autologous cell , xenogeneic cell (para. 143), allogenic transplantation (para. 3) and adoptive immunotherapy " (para. 238). All of the contemplated cell therapies are for the treatment of HIV or AIDS and thus the skilled artisan would find that the claimed method of introducing any CCR5 and B2M gene edited cell or a population of cells to a subject in need thereof thus would encompass treating said subject having HIV or AIDS.
It is noted that although instant claims have been amended to recite a method of administering cells to a subject in need of such cells, comprising (a) altering any target polynucleotide sequence associated with a broad class of disorder in a cell or a population of cells ex vivo (b) altering a target B2M polynucleotide sequence in the cell or population of cells ex vivo by contacting the target B2M polynucleotide sequence with a Cas protein and at least one ribonucleic acid and administering the cell or population of cells via any route into the subject in need thereof such cell, they have been analyzed for their intended use in the subject in need thereof suffering from, being treated for, diagnosed with, suspected of having, or at increased risk of developing HIV or AIDS. The cells can also be obtained from a normal healthy subject not suffering from, being treated for, diagnosed, suspected of having, or at increased risk of developing, the disorder (see para. 104 of the specification). The present invention contemplates genomically editing any primary human cells to cleave B2M gene sequences, as well as editing the genome of such cells to alter target CCR5 polynucleotide sequences. The disclosure provided by the applicant, in view of prior art, must encompass a wide area of knowledge to a reasonably comprehensive extent. In other word each of these, aspect must be shown to a reasonable extent so that one of the ordinary skills in the art would be able to practice the invention without any undue burden being on such Artisan.
Guidance of the Specification and The Existence of Working Examples:
The specification teaches designing gRNAs to target Cas9 to the B2M gene (FIG. 5A). Each guide was first tested for the ability to direct site-specific mutations in HEK293T cells. Using flow cytometry, we measured the efficiency of each gRNA to direct Cas9-mediated ablation of B2M surface expression 72 hours post-transfection (FIG. 5B). We observed that B2M was abrogated in-7% (1.02 SEM, n=3) to 48% (.+-.80 SEM, n=3) of HEK293T cells depending upon the gRNA utilized (FIG. 5C and FIG. 6A). Similar results were observed using the Surveyor assay, with gRNA-specific mutation frequencies of 0-26% in HEK293T cells (FIG. 6B). It is further disclosed that variation in the efficiency with which a specific gRNA directed Cas9- mediated ablation was observed, even between gRNAs targeting the same exon or nearly overlapping sites (FIG. 5A-E) indicating that on-target efficiency of site directed mutation is highly gRNA dependent as previously noted by Hsu et al (Nat Biotechnology. 2013 Sep;31(9):827-32). The specification further teaches that the activity of the CRISPR/Cas9 system .It is remarkably variable in different human cell types, with the same gRNA exhibiting highly efficacious on target mutagenic activity in HEK293T cells but little activity in CD4+ T cells (emphasis added). In contrast, the targeting efficacy in K562 cells and CD34+ HSPCs was comparable. Moreover, consistent with previous reports (Hsu et al., 2013) the specification discloses that the efficiency of the CRISPR/Cas9 system was gRNA specific, as even gRNAs with partially overlapping sequences within the same exon displayed significantly different targeting efficiencies.
Further, a dual gRNA approach yielded increased gene ablation efficacy in both CD4..+ T cells and CD34.sup.+ HSPCs leading to predicted deletions at the targeted loci (see para. 247). Figure 9A and 9B demonstrate potential off-target sites identified in CCRS5 homologue CCR2 and analysis of events detected at the single off-target site in which mutagenesis was significantly detected above background. Figures 10A, 10B, 10C and 10D demonstrate that CCR5-edited CD34+ HSPCs retain multi-lineage potential. The specification teaches transplanting CD34+HPSC expressing Cas9 with crCCR5 gRNA into NSG mouse (immunodeficient mouse) and characterization of HPSC chimerism is analyzed Representative FACS plot showing human hematopoietic cell (hCD45+) engraftment and multi-lineage reconstitution at 12 weeks post-transplantation in the bone marrow (FIG. 10B) and spleen (FIG. 10C) of NSG recipient mice. The specification is completely silent on providing any guidance on a method of administering any cell to any subject having HIV or AIDS intended for treating HIV infection and/or treatment of AIDS, using any other Cas protein, gRNA targeting CCR5 and B2M or using any other genetically modified cells as broadly claimed in the instant application (emphasis added).
State of the Art and Predictability of the Art and the Amount of Experimentation Necessary:
The claimed method encompasses administering any cell or any population of cells to a subject in need thereof to a subject, said method comprising altering a target CCR5 polynucleotide sequence in a cell or a population of cells ex vivo by contacting the CCR5 polynucleotide sequence, wherein the subject has HIV or AIDS. However, the specification provides no examples of administering any genetically modified cells or the population of cells for the treatment of any HIV infection of different subtype (HIV-1 or HIV-2) of different etiology and pathology using the cells of the invention that would enable the method as claimed.
A Lack of Nexus between Administration and Treatment or Prevention
The breadth of disease encompasses delivering any genetically edited cells (B2M and CCR5) to any subject having HIV or AIDS. However, a review of the instantly filed specification and the art, the skilled artisan would find that administering the genetically modified cells or population of cells of the claimed method for the purposes of treating or preventing would not treat HIV or AIDS. In a post filing art Wang (Frontiers in Immunology, 2025, 16, 1-8) states “effective CCR5 disruption, HIV switches coreceptor usage to CXCR4 (X4-tropic strains), enabling continued infection. Furthermore, once HIV integrates into the host genome, the virus can be reactivated via the long terminal repeat (LTR) region, which contains strong promoter and enhancer elements. This allows viral reactivation even in cells lacking CCR5 or CXCR4 expression. Activation of LTR promotes Gag expression, enhances viral particle assembly, and facilitates reverse transcription, followed by integrase-mediated insertion of viral DNA into host chromosomes, ultimately driving viral replication” (see page 03, col. 1, para. 2, section 3.1 and references therein). In another post filing art summarized by the reference of Xu et al (NEJM, 2019, 381, 1240-1247) reported “successful transplantation and long-term engraftment of CRISPR-edited HSPCs, the percentage of CCR5 disruption in lymphocytes was only approximately 5%, which indicates the need for further research into this approach” (abstract). In fact, Xu emphasizes “It will be necessary to analyze the CRISPR–Cas9–mediated CCR5 ablation in HSPCs further under a higher gene-targeting efficiency.” (see page 1246, col. 1, last para. to col. 2). In the instant case, neither prior art nor instant specification provides no guidance with respect to administering any genetically modified cells edited for both B2M and CCR5 in any subject intended to treat HIV or AIDS in any subject. Therefore, the skilled artisan would find that such a breadth would be unpredictable given the teachings of the specification, the breadth of cells to be administered and the lack of teachings in the art that would support the breadth of the claimed invention. Further, instant specification provides any nexus recited in the claims between "introducing a genetically modified gene edited cell or a genetically modified cells to the subject that has HIV or AIDS" that results in preventing or treating HIV infection or AIDS in subject. The claimed method has operable step that is the administration of the genetically modified gene edited cells There is no recitation of how the subject is being treated or provided any treatment or prevention of HIV or AIDS encompassed by the breadth of the claim by the administration of said genus of gene edited cells.
Unpredictability of multiplex gene editing
The claims encompass (a) altering a CCR5 target polynucleotide sequence in any cell or a population of cells ex vivo by contacting the polynucleotide sequence with any Cas protein and at least one ribonucleic acid, wherein the ribonucleic acid directs Cas protein to and hybridizes to any CCR5 target motif of the target polynucleotide sequence, wherein the CCR5 target polynucleotide sequence is cleaved; (b) altering a target B2M polynucleotide sequence in the cell or population of cells ex vivo by contacting the target B2M polynucleotide sequence with a Cas protein and at least one ribonucleic acid selected from the group consisting of SEQ ID NOs: 9-23 and 419- 2609.
The state of the art before and after filing of instant application teaches multiplex gene editing significantly increases the risk of unintended chromosomal deletions and translocations compared to making a single edit. In this regard, Aussel et al (Nature Communication, 2025, 7208, 1-5 and references therein, art of record) teaches CRISPR/Cas system can also induce other SVs, including translocation between homologous chromosomes that results in an acentric and a dicentric chromosome, large deletions following two cleavage events on the same chromosome, and translocations between two different (heterologous) chromosomes, e.g. upon simultaneous cleavage of the target site and an OT site (see Frock, et al. Nat. Biotechnol. 33, 179-186, 2015). As with any type of genomic aberration, from point mutations to large-scale chromosomal rearrangements, can ultimately lead to hazardous cellular consequences (see page 1, col. 2, last para to page 2, col. 1, para. 1 and Hunt et al Hum Genet 2023 Apr 24;142(6):705-720, table 2). Kalter et al (Mol Ther Nucleic Acids 2025 Jul 17;36(3):102636 and references therein, art of record) teaches that while CRISPR-Cas system is highly efficient and versatile, adverse editing events at unintended sites, known as off-target activity (OTA), remains a significant safety concern. OTA can occur when the CRISPR-Cas complex associates genomic loci with high sequence identity to the on-target sites, and is further complicated by the tolerance of DNA and RNA base bulges by CRISPR-Cas complexes (Figure 3A). In the instant case, claims are broadly requiring altering CCR5 target polynucleotide sequence by contacting the polynucleotide sequence with any Cas protein and at least one ribonucleic acid, wherein the ribonucleic acid directs Cas protein to and hybridizes to any target motif of the CCR5 target polynucleotide sequence. The guidance provided in the specification is limited to CCR5- edited CD34+ HSPCs using specific gRNA as set forth in Fig. 2 and Cas9. Ellwanger et al (Virus Research, 2020, 286, 198040, 1-24) in a post filing publication state "participation of CCR5 in viral infections is complex and varied and, therefore, cannot be generalized. This article also pointed out neglected gaps in knowledge involving CCR5 that should be addressed in future studies" (see page 16, col. 2, last para.). The study of Ellwanger clearly shows that targeting CCR5 to treat or prevent viral infection was not predictable and neither prior art or instant specification provide any guidance to provide art recognized unpredictability.
The independent claim recite altering a target CCR5 and B2M polynucleotide sequence in any cell or any population of cells ex vivo. As stated before, the specification teaches that the activity of the CRISPR/Cas9 system is remarkably variable in different human cell types, with the same gRNA exhibiting highly efficacious on target mutagenic activity in HEK293T cells but little activity in CD4+ T cells. In contrast, the targeting efficacy in K562 cells and CD34+ HSPCs was comparable. Moreover, consistent with previous reports (Hsu et al., 2013) the specification discloses that the efficiency of the CRISPR/Cas9 system was gRNA specific, as even gRNAs with partially overlapping sequences within the same exon displayed significantly different targeting efficiencies. Further, a dual gRNA approach yielded increased gene ablation efficacy in both CD4..+ T cells and CD34.sup.+ HSPCs leading to predicted deletions at the targeted loci (see para. 247). The ribonucleic acid sequences of all gRNA, other than SEQ ID NO: 16 and 21 for B2M and SEQ ID NO: 2 to 8 for CCR5 encompassed within the genus of ribonucleic acid sequences have not been disclosed to have contemplated biological activity in any human primary cells other than CD34+ cells. Based upon the prior art there is expected to be variation among the species of gRNA sequences depending upon the species of primary cells. The specification has provided the description of use of dual gRNA approach yielding increased gene ablation efficacy in both CD4+ and T cells for the claimed genus of gRNA encompassed by the claims. The art indicated that there is variation between use of gRNA sequences and gene deletion efficiency in different human cells. It is noted that claims also recite use of any Cas protein subsequently limiting to Cas comprising a Cpfl protein. The specification fails to provide any guidance with respect to use of Cpfl protein that would work in genus of primary human cells. Before the effective filing date of instant invention, Zetsche et al (Cell, 2015, 163, 759-771, 09/25/2015) tested the activity of each Cpf1-family protein, we selected a guide RNA target site within the DNMT1 gene (Figure 7B). It was noted that each of the Cpf1- family proteins along with its respective crRNA designed to target DNMT1 was able to cleave a PCR amplicon of the DNMT1 genomic region in vitro ( Figure 7 C). However, when tested in human embryonic kidney 293FT (HEK293FT) cells, only two out of the eight Cpfl-family proteins (7, AsCpfl and 13, LbCpf1) exhibited detectable levels of nuclease-induced indels (figures 7 C and 7D). The claimed invention as a whole is not adequately described if the claims require essential or critical elements or motifs which are not adequately described in the specification and which is not conventional in the art as of applicants effective filing date. An artisan would have to perform undue experimentation to make and use the invention, without reasonable expectation of success. The art further teaches that cells with B2M-KO lack Major Histocompatibility Complex (MHC) class I on their surface avoid detection and killing by cytotoxic T cells, which typically target cells presenting antigens via MHC-I. However, the art teaches NK cells act as a backup surveillance system and possess a mechanism called "missing- self" recognition. In this regard, Sun teaches B2m-/- cells are rapidly rejected by wild-type NK cells in chimeric mice following viral infection (Fig. 4a-b). (Sun et al J Immunol. 2008 Dec 1;181(11):7453-7457.) It is known in art that B2M is component of MHC class I complex that helps the immune system's T cells to recognize cancer cells. However, art recognizes that editing B2M gene is also expected to evade the host immune system that may contribute to resistance to standard immunotherapies as evident from the teaching of Han (Frontiers of Immunology, 2025, 16:1512509, 1-15, and references therein, see page 4, col. 2, para. 1). In view of foregoing, it is apparent that there is no nexus between the in vitro examples of producing genetically modified CD34+ HSC or T cells of the invention and the in vivo delivery of genetically modified cell or population of cell that is provided by administering said genetically modified cells as claimed via any route to treat or prevent a subject having HIV or AIDS. An artisan would have to perform undue experimentation to make and use the invention, without reasonable expectation of success.
Unpredictability of introducing cell or population of cells into the subject
The claims encompass administering the genetically altered cell to a subject in need thereof intended to treat a subject that has HIV or AIDS. The claimed invention relies upon targeting of the genetically modified cells to the breadth of species that encompass cancer. There are no teachings in the specification that would teach or suggest that the genetically modified cells or population of cells, such as any somatic cell, hepatocyte or pluripotent cells would be able to treat a subject that has HIV or AIDS. Again, there is no nexus between the in vitro examples of producing genetically modified cells that are edited for B2M or CCR5 the in vivo therapy that is provided by administering said genetically modified cells in a subject having HIV or AIDS . The claims encompass treating or preventing a subject having HIV or AIDS by administering via any route the recited genetically modified cell or population of cells. However, in view of the teachings of prior art, there would be significant unpredictability regarding just delivering somatic cells to the subject having HIV or AIDS. Further, there exists no nexus, by which administered somatic cells can actually treat HIV or AIDS and neither the art or the specification provides any mechanism of action by which the administered genetically modified somatic cells would provide any treatment or therapeutic effect to the breadth of transplanting such cells that would enable the claimed method. The claims encompass xenogeneic transplantation of genetically modified cell or population of cells. The specification fails to provide any predictable guidance as to how the genetically modified cell or population of cell from one subject that is edited for B2M and any region of CCR5 target polynucleotide sequence would be engrafted in another subject as required by the method of treating or preventing a disorder associated with CCR5 expression. For instance, Frederiksen (Front. Genome Ed. 2024, 6:1403395. 1-17 and references therein) teaches transplantation of immune-evasive hESCs (B2M-/-) into immune competent BALB/c mice resulted in complete rejection within 11 days, with clear immune infiltration of T-cells on day 8. The results reveal that knockout of B2M and CIITA together with species specific expression of CD47 are insufficient to prevent rejection in an immune competent and xenogeneic context (page 1, last para.). An artisan would have to perform undue experimentation to make and use the invention, without reasonable expectation of success.
In conclusion, in view of breadth of the claims and absence of a strong showing by Applicant, in the way of specific guidance and direction, and/or working examples demonstrating the same, such invention as claimed by Applicant is not enabled for the claimed inventions. An artisan of skill would have required undue experimentation to practice the invention, without reasonable expectation of success as supported by the observations in the art record.
Response to arguments
Applicant disagree with the rejection arguing that the specification describes the use of the CRISPR/Cas9 system to target two clinically relevant genes, B2M and CCR5. See, e.g., as-filed application at paragraph [0238]. CCR5 is "the main co-receptor used by CCR5-tropic strains of HIV-1 (Trkola et al., 1996) and a validated target for gene ablation, as mutations resulting in loss of protein expression or haploinsufficiency protect against HIV infection [0239] and FIG. 5. Thus, the application enables the targeting of a CCR5 polynucleotide sequence with a Cas protein and at least one ribonucleic acid. In addition, the claimed method further recites altering a target B2M polynucleotide sequence ex vivo by contacting the B2M sequence with a Cas protein and at least one ribonucleic acid selected from the group consisting of SEQ ID NOs: 9-23 and 419-2609. The application teaches the use of single gRNAs to target B2M. See id. at paragraph [0240] and FIGS. 5 and 6. The modified cells may then be transplanted or administered into a subject, e.g., a mouse. See id. at paragraph [0244]. Applicants’ arguments have been fully considered, but are not found persuasive.
In response, it is noted that office action explicitly indicated that instant specification enables a method of engrafting an allogeneic CD34+ hematopoietic stem cells to a mouse in need, the method comprising:
(a) altering a target CCR5 polynucleotide sequence in a population of CD34+ hematopoietic stem cells ex vivo by contacting the CCR5 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated-9 (Cas 9) protein and at least one ribonucleic acid selected from group consisting of SEQ ID: 2-8, wherein the ribonucleic acid directs Cas protein to and hybridizes to a target motif of the target CCR5 polynucleotide sequence,
(b) altering a target B2M polynucleotide sequence in the population of CD34+ hematopoietic stem cells ex vivo by contacting the target B2M polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated-9 (Cas9) protein and at least one ribonucleic acid selected from the group consisting of SEQ ID NOs: 16 or 21 and 419-2609 to obtain a population of genetically modified CD34+ HSC comprising a genome in which B2M and CCR5 gene has been cleaved thereby eliminating surface expression of MHC1 molecule in the population of CD34+ hematopoietic stem cells; and
(c) administering the allogeneic population of genetically modified CD34+ hematopoietic stem comprising a genome in which B2M and CCR5 gene have been edited into the mouse, thereby engrafting CD34+ HSC in the subject in need thereof. The specification is silent on altering genus of target CCR5 and B2M polynucleotide sequence in any other cell specie or a population of cell species ex vivo, using different class of a Cas protein to any other subject having HIV or AIDS for the reasons discussed above in the body of the rejection. The guidance provided in the specification is limited to transplanting CD34+HPSC expressing Cas9 with crCCR5 gRNA into NSG mouse (immunodeficient mouse) and characterization of HPSC chimerism. There is no evidence on transplanting any cells including CD34+HPSC or T cells expressing Cas9 with crCCR5 gRNA and B2M gRNA in a subject having HIV or AIDS. Therefore, transplanting CD34+HPSC (a specific cell type) expressing Cas9 (specific Cas protein) with crCCR5 gRNA (one target gene) into NSG mouse (immunodeficient mouse that is not a model animal) for the characterization of HPSC chimerism does not provide reasonable correlation of a method of transplanting any cell or any population of cells that is genetically edited for B2M and CCR5 using plurality of gRNA and Cas protein in any subject having HIV or AIDS intended to treat or prevent the disorder as broadly claimed. An artisan would have to perform undue experimentation to make and use the invention, without reasonable expectation of success for the reasons discussed above in the body of the rejection.
Therefore, in view of the fact patterns of the instant case, and the ground of rejection outlined by the examiner, applicants' arguments are not compelling and do not overcome the rejection of record.
Withdrawn-Claim Rejections - 35 USC § 112
Claims 50-60 were rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. Applicant’s amendments to the base claim obviates the basis of the rejection.
New-Claim Rejections - 35 USC § 112- necessitated by amendments
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 61 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 61 recites the limitation "the altered cells " in line 1 There is insufficient antecedent basis for this limitation in the claim. It is unclear if the altered cell is produced after step (a) or (a) and (b). Appropriate corrected is required.
New-Claim Rejections - 35 USC § 103- necessitated by amendments
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 50, 53-54, 56-60 and 61 are rejected under 35 U.S.C. 103 as being unpatentable over Musunuru et al (WO2014/165825, dated 10/09/2014) as evidenced by Gussow et al (J. Immunol. 139 (9), 3132-3138, 1987, IDS) and NCBI accession no NG_012920.1 (07/08/2013, pages 1-6, IDS)/ Naldini et al (WO2016/063264, filed 10/23/15, EFD 10/24/2014 < IDS or USPGPUB 20190032049),
Claim interpretation: Instant rejection is applied to the breadth of the active methos steps and not necessarily to a method intended for treating a subject having HIV or AIDS by administering the edited cells. Additionally, altering a target CCR5 polynucleotide and a target B2M polynucleotide sequence is interpreted to be simultaneously in the same cell.
Claims are directed to a method of administering cells to a subject in need of such cells, the method comprising: (a) altering a target CCR5 polynucleotide sequence in a cell or a population of cells ex vivo by contacting the CCR5 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and at least one ribonucleic acid, wherein the ribonucleic acid directs Cas protein to and hybridizes to a target motif of the target CCR5 polynucleotide sequence, wherein the target polynucleotide sequence is cleaved;(b) altering a target B2M polynucleotide sequence in the cell or population of cells ex vivo by contacting the target B2M polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and at least one ribonucleic acid selected from the group consisting of SEQ ID NOs: 9-23 and 419-2609; and(c) administering the cell or population of cells into the subject, wherein the subject has HIV or AIDs.
With respect to claim 50, Musunuru teaches a method of administering cells that
has a reduced likelihood of triggering a host immune response that utilizes two or more ribonucleic acids which guide Cas protein to and hybridize to a target polynucleotide sequence efficiently and effectively deletes target polynucleotide sequences that includes CCR5 and/or B2M in human CD34+ and T cells that is administered to a subject in need thereof (see para. 113). Musunuru teaches contacting a polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and/or ribonucleic acids is intended to include incubating the Cas protein and/or the ribonucleic acids in the cell together in vitro or ex vivo (see para. 116, 121). It is further disclosed that target polynucleotide sequence is a variant, a homolog or an ortholog of CCR5 (see para. 139, 176). Musunuru teaches contacting a cell or population of cells ex vivo with (i) a Cas protein, (ii) at least two ribonucleic acids which direct Cas protein to and hybridize to a target polynucleotide sequence encoding B2M in the cell or population of cells, wherein the target polynucleotide sequence encoding B2M in the cell or population of cells is cleaved, thereby reducing the likelihood that the cell or population of cells will trigger a host immune response in the subject, (iii) at least two additional ribonucleic acids as set forth in SEQ ID NO: 301 which direct Cas protein to and hybridize to a CCR5 target polynucleotide sequence in the cell or population of cells, wherein the CCR5 target polynucleotide sequence is cleaved and (b ) administering the resulting cell or cells containing CCR5 and B2M edited cells to a subject in need of such cells (see claims 316 and 317).
With respect to claim 53, Musunuru teaches that the method further comprising altering one or more additional polynucleotide sequences in the cell ex vivo.(see para. 7, 8).
Regarding claim 54, Musunuru teaches the method, wherein the Cas protein comprises a Cas9 protein (see para. 201, 212).
With respect to claim 56, Musunuru teaches that the cell or population of cells are selected from the group consisting of a stem cell, a CD34+ hematopoietic progenitor cells (HPCs), hepatocyte, and primary CD4+ T cells (see para. 18, 307),
Regarding claim 57. Musunuru teaches that method, wherein the alteration of the target B2M polynucleotide sequence in the cell or population of cells results in the deletion of a contiguous stretch of genomic DNA, thereby eliminating surface expression of MHC Class I molecules in the cell or population of cells (see para. 136, 315, fig. 4D).
With respect to claim 59-60, Musunuru teaches the cell or population of cells are CD4+ T cells or CD34+ cells.
Regarding claim 61, Musunuru teaches that the method that are subsequently contacted with an agent or growth factor differentiation agent to differentiate the cells further (see para. 116). Musunuru differs from claimed invention by not disclosing that the at least one ribonucleic acid is selected from group consisting of SEQ ID NO: 16 and 21.
Before, the effective filing date of instant application, Naldini et al et al teach human HEK-293 T cell or a population of human HEK-293 T cells (page 84, line 5) that is transfected with different B2M gRNA together with Cas9 (see page 84, lines 5-8, table 4) to produce B2M edited human HEK-293 T cells. It is relevant to noted that at least one of the ribonucleic acid sequences as set forth in SEQ ID NO 7 disclosed by Naldini has 100% sequence identity to claimed sequence as set forth in SEQ ID NO: 21 (table 4) meeting the limitation of the claims. Naldini teaches that cells may be a human primary T cell (example 12, 16, page 28), stem cell or progenitor cells (see page 10, lines 11-14, page 19, last line to page 20, lines 1-2). Gussow or NG_012920.1 discloses a nucleic acid sequence that is 100% identical to SEQ ID NO: 16, identified as Homo sapiens beta2microglobulin (B2M), on chromosome 15 and 100% identical to SEQ ID NO: 21 of the instant application to nucleic acids 5093-5112 of the sequence of accession NG_012920 identified as Homo sapiens beta2microglobulin (B2M), on chromosome 15).
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art seeking to administer cells to a subject in need of such cells would combine the teaching of prior art to modify the method of Musunuru by including at least one ribonucleic acids corresponding to the DNA sequences SEQ ID NO: 16 and SEQ ID NO: 21 , as previously disclosed by Gussow and NCBI accession no/ Naldini, to have specifically targeted a region of the B2M gene using as guide RNAs, as instantly claimed, with a reasonable expectation of success, before the effective filing date of the instant application, to provide a primary cells having dual edited cells (B2M and CCR5), thereby eliminating surface expression of MHC Class I molecules. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would be motivated to do so because a deletion mutant without a continuing open reading frame that would have resulted in the abrogation of the formation of a mature B2M mRNA, thereby eliminating surface expression of MHC Class I molecules in the cell or population of cells. One of skill in the art would have had a reasonable expectation of success in the abrogating the formation of a mature B2M mRNA and CCR5 in primary human cells or a population of human primary cells because prior art successfully reported use of Cas9 mRNA and sgRNAs to disrupt B2M and CCR5 in a cell as evident from the teaching of Musunuru (see Fig. 4A-D). It should be noted that the KSR case forecloses the argument that a specific teaching, suggestion, or motivation is required to support a finding of obviousness See the recent Board decision Ex parte Smith, --USPQ2d—, slip op. at 20, (Bd. Pat. App. & Interf. June 25, 2007) (citing KSR, 82 USPQ2d at 1396) (www.uspto.gov/web/offices/dcom/bpai/prec/fd071925.pdf).
Claim 55 is rejected under 35 U.S.C. 103 as being unpatentable over Musunuru et al (WO2014/165825, dated 10/09/2014) as evidenced by Gussow et al (J. Immunol. 139 (9), 3132-3138, 1987) and NCBI accession no NG_012920.1 (07/08/2013, pages 1-6)/ Naldini et al (WO2016/063264, filed 10/23/2015, EFD 10/24/2014 or USPGPUB 20190032049) as applied above for claim 50, and further in view of Zetsche et al (Cell, 2015, 163, 759-771, 09/25/2015).
The teaching of Musunuru , Gussow , accession no/ Naldini have been described above and relied in same manner here. Regarding claim 61, Musunuru teaches that the method that are subsequently contacted with an agent or growth factor differentiation agent to differentiate the cells further (see para. 116). The combination of references differs from claimed invention by not disclosing that Cas protein comprises a Cpf1 protein.
However, before, the effective filing date of instant application, Zetsche et al reported characterization of Cpf1, a putative class 2 CRISPR effector. Zetsche et al demonstrated that Cpf1mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break (abstract). Zetsche et al disclose advantage of using Cpf1 because unlike Cas9 systems, Cpf1-containing CRISPR systems have three features. First, Cpf1-associated CRISPR arrays are processed into mature crRNAs without the requirement of an additional trans-activating crRNA (tracrRNA). Second, Cpf1-crRNA complexes efficiently cleave target DNA proceeded by a short T-rich protospacer-adjacent motif (PAM), in contrast to the G-rich PAM following the target DNA for Cas9 systems. Third, Cpf1 introduces a staggered DNA double stranded break with a 4 or 5-nt 50 overhang (see page 760, col. 1, last para.).
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art seeking to administer cells to a subject in need of such cells would combine the teaching of prior art to modify the method of Musunuru and accession no/Naldini by substituting Cas-mediated genomic editing systems al with functionally equivalent Cpf1 as disclosed by Zetsche, with a reasonable expectation of success, before the effective filing date of the instant application, in order to more efficiently produce allogenic disorder-specific donor cells from primary cells isolated from a subject in a single step. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would be motivated to do so because prior art explicitly suggested that Cpf1 generates a staggered cut with a 50 overhang, in contrast to the blunt ends generated by Cas9. This structure of the cleavage product could be particularly advantageous for facilitating non-homologous end joining (NHEJ)-based gene insertion into the mammalian genome (see Zetsche page 765, col. 1, para. 2). One of skill in the art would have had a reasonable expectation of success in using Cpf1 for abrogating the formation of a mature mRNA of a target gene in primary cells because prior art successfully reported use of Cpf1 for genome editing in mammalian cells (see figure 7). It should be noted that the KSR case forecloses the argument that a specific teaching, suggestion, or motivation is required to support a finding of obviousness See the recent Board decision Ex parte Smith, --USPQ2d—, slip op. at 20, (Bd. Pat. App. & Interf. June 25, 2007) (citing KSR, 82 USPQ2d at 1396) (www.uspto.gov/web/offices/dcom/bpai/prec/fd071925.pdf).
Claims 50, 53-60 and 61 are rejected under 35 U.S.C. 103 as being unpatentable over Musunuru et al (WO2014/165825, dated 10/09/2014), Meissner et al (Methods in Enzymology, 2014, Volume 546 chapter 13, 273-293)/Mandal et al (Cell 2014, 15, 643-652) as evidenced by Naldini et al (WO2016/063264, file 10/23/2015, EFD 10/24/2014 or USPGPUB 20190032049).
The teaching of Musunuru , Gussow , accession no/ Naldini have been described above and relied in same manner here. Regarding claim 61, Musunuru teaches that the method that are subsequently contacted with an agent or growth factor differentiation agent to differentiate the cells further (see para. 116).
With respect to claims 50, 53-54, 56, 58-59, Meissner et al teach a method of altering an isolated primary human CD4+ T cells by contacting a Cas protein in combination with two guide sequences similar to one disclosed in the instant application targeting B2M (see page 275, para. 3, page 276, last paragraph; figure 13.1, 13.5) to produce human CD4+ T whose genome comprises a deletion of a 2.2 kb of contiguous stretch of genomic DNA of the beta2-microglobulin gene resulting in lack of surface expression of MHC molecule (see 275, para. 3).
Regarding claims 50, 53-54. 56, 60, Meissner further teaches altering CD34+ HSPCs using the CRISPR/Cas9 system by contacting cells with Cas9 and pair of gRNAs targeting CCR5 to produce cells are deficient in CCR5 gene (see fig. 13.7).
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Regarding claim 50, Meissner reported targeting CCR5 in HSPCs represents an attractive way of creating an HIV-1-resistant immune system. It is disclosed that upon transplantation, CCR5-deficient HSPCs can engraft, expand and ultimately give rise to CCR5-deficient CD4+ T cells that are resistant to HIV-1 virus infection (see page 275, last para. to page 276, para. 1). With respect to claim 27, Meissner et al teaches generating universally transferable donor T cells that are readily available and can be used in an allogeneic transplantation setting and thus be administered to multiple recipients with disparate MHC expression for a variety of disorder (see page 275, para. 4). Likewise, Mandal teaches teach a method of altering an isolated primary human CD4+ T and CD34+ HPC cells by contacting a Cas protein in combination with guide sequences as set forth in SEQ ID NO: 16 and/or 21 targeting B2M (see fig. 1 and 2 and supplementary data ) to produce human cells whose genome comprises a deletion of contiguous stretch of genomic DNA of the beta2-microglobulin gene resulting in lack of surface expression of MHC molecule (see 275, para. 3). Regarding claims 50, 53-54. 56, 60, Mandal further teaches altering CD34+ HSPCs or using the CRISPR/Cas9 system by contacting cells with Cas9 and pair of gRNAs targeting CCR5 to produce cells are deficient in CCR5 gene (see fig. 3-4) Mandal further contemplate that CRISPR/Cas9 can be used to ablate genes of clinical significance in CD4+ T cells and CD34+ HSPCs with an efficiency that is therapeutically meaningful for a number of clinical settings, such as the treatment of HIV (see page 650. Col. 1, last para.)
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art seeking to administer cells to a subject in need of such cells would combine the teaching of prior art to modify the method of Musunuru in view of Meissner/Mandal by including at least one ribonucleic acids corresponding to the DNA sequences SEQ ID NO: 16 and SEQ ID NO: 21 , as previously disclosed by Gussow and NCBI accession no/ Naldini, to have specifically targeted a region of the B2M and CCR5 gene using as guide RNAs, as instantly claimed, with a reasonable expectation of success, before the effective filing date of the instant application, to provide a primary cells containing B2M and CCR5 edited cells, thereby eliminating surface expression of MHC Class I molecules and conferring resistance to HIV infection. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would be motivated to do so because a deletion mutant that would have resulted in the abrogation of the formation of a mature B2M and CCR%, thereby eliminating surface expression of MHC Class I molecules in the cell or population of cells to produce MHC Class I negative cells. One of skill in the art would have had a reasonable expectation of success in the abrogating the formation of a mature B2M and CCR5 in primary human cells or a population of human primary cells because prior art successfully reported use of Cas9 mRNA and sgRNAs to disrupt B2M and CCR5 in a cell as evident from the teaching of Musunuru and Meissner/Mandal (see above). It should be noted that the KSR case forecloses the argument that a specific teaching, suggestion, or motivation is required to support a finding of obviousness See the recent Board decision Ex parte Smith, --USPQ2d—, slip op. at 20, (Bd. Pat. App. & Interf. June 25, 2007) (citing KSR, 82 USPQ2d at 1396) (www.uspto.gov/web/offices/dcom/bpai/prec/fd071925.pdf).
Conclusion
No claims allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Riolobos et al (Mol. Ther 2013 Jun; 21(6):1232-41) , disclose a human embryonic stem cell line or population of human ES cells (abstract) comprising a genome in which the B2-microglobulin (B2M) gene on chromosome 15 has been edited to delete a contiguous stretch of genomic DNA (abstract, page 1234, col. 2, para. 2 - page 1235, col. 2, para 1 ), thereby eliminating surface expression of MHC Class I molecules in the cell or population of cells (page 1234, col. 2, para. 2 - page 1235, col. 2, para. 1; page 1233, col. 1, para. 2). Riolobos et al further disclose allogenic transplantation of cells including administration of said cells (page 1232, col. 1 -col 2, para. 2; page 1233, col. 1, para. 2).
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/ANOOP K SINGH/ Primary Examiner, Art Unit 1632
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